1. Field of the Invention
The present invention relates to methods for the treatment or prevention of disease states induced by activation of the A3 receptor and mast cell activation.
2. Discussion of the Related Art
Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular nervous, pulmonary, renal and immune systems. Adenosine produces bronchoconstriction in asthmatics but not in nonasthmatics by triggering mast cell degranulation (Cushly et al., 1984, Am. Rev. Respir. Dis. 129:380-384). This suggests that the activation of an adenosine receptor contributes to bronchoconstriction in asthmatics.
The amino acid sequence for the human A3 receptor is 72% identical with the rat A3 receptor and 85% identical with the sheep A3 receptor sequences.
The actions of adenosine are mediated through G-protein coupled receptors, the A1, A2A, A2B and A3 adenosine receptors. The degranulation of rat mast cells has been attributed to the activation of A3 receptors (Ramkumar V, Stiles G L, Beaven M A and Ali H (1993), "The A3 Adenosine Receptor Is the Unique Adenosine Receptor Which Facilitates Release of Allergic Mediators in Mast Cells," J Biol Chem 268(23):16887-216890). The adenosine receptors were initially classified into A1 and A2 subtypes on the basis of pharmacological criteria and coupling to adenylate cyclase (Van Caulker, D., Muller, M. and Hamprecht, B. (1979) J. Neurochem. 33, 999-1003.). Further pharmacological classification of adenosine receptors prompted subdivision of the A2 class into A2A and A2B subtypes on the basis of high and low affinity, respectively, for adenosine and the agonists NECA and CGS-21680 (Bruns, R. F., Lu, G. H. and Pugsley, T. A. (1986) Mol. Pharmacol. 29, 331-346; Wan, W., Sutherland, G. R. and Geiger, J. D. (1990) J. Neurochem. 55, 17631771). The existence of A1, A2A and A2B subtypes has been confirmed by cloning and functional characterization of expressed bovine, canine, rat and human receptors.
A fourth subtype, A3, had remained pharmacologically undetected until its recent identification by molecular cloning. The rat A3 sequence, tgpcrl, was first cloned from rat testis by Meyerhoff et al. (Meyerhof W, Muller-Brechlin R and Richter D (1991) Molecular Cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis. Febs Lett 284:155-160.). Subsequently, a cDNA encoding the identical rat receptor was cloned from striatum and functionally expressed by Zhou et al. (Zhou Q Y, Li C, Olah M E, Stiles G L and Civelli O (1992), "Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor," Proc Natl Acad Sci USA 89:7432-7436.). When compared to the other members of the G-protein coupled receptor family, the rat sequence had the highest homology with the adenosine receptors (&gt;40% overall identity, 58% within the transmembrane regions). When stably expressed in CHO cells, the receptor was found to bind the radioligand .sup.125 I-APNEA (N.sup.6 -2-(4-amino-3-iodophenyl)ethyladenosine) and when transfected cells were treated with adenosine agonists, cyclic AMP accumulation was inhibited with a potency order of NECA=R-PIA&gt;CGS21680. The rat A3 receptor exhibited a unique pharmacology relative to the A1 and A2 adenosine receptor subtypes and was reported not to bind the xanthine antagonists 1,3-dipropyl-8-phenylxanthine (DPCPX) and xanthine amine congener (XAC). Messenger RNA for the rat A3 adenosine receptor is also primarily expressed in the testis.
The sheep homolog of the A3 receptor was cloned from hypophysial pars tuberalis (Linden J, Taylor H E, Robeva A S, Tucker A L, Stehle J H, Rivkees S A, Fink J S and Reppert S M (1993). "Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution," Mol Pharmacol 44:524-532.). The sheep receptor is 72% identical to the rat receptor, binds the radioligand .sup.125 I-ABA and is coupled to inhibition of cyclic AMP. The agonist affinity order of the sheep receptor is I-ABA&gt;APNEA&gt;NECA.gtoreq.R-PIA&gt;&gt;CPA. The pharmacology of xanthine antagonists was extensively studied and, contrary to what had been found in the rat, the sheep receptor was found to exhibit high affinity for 8-phenylxanthines with para-acidic substitutions. Also, in contrast to the rat where transcript is primarily located in testis, the transcript for the sheep A3 adenosine receptor is widespread throughout the brain and is most abundant in the lung and spleen. In sheep moderate amounts of transcript are also observed in pineal and testis. Thus, because the published literature provides an inconsistent profile of adenosine A3 receptor pharmacology and tissue distribution, it was not possible to predict the pharmacology or tissue distribution of the human A3 adenosine receptor.
Based on the use of tissue rich in particular adenosine receptor subtypes, assays have been described to identify adenosine receptor agonists and antagonists and determine their binding affinity (see GB 2 264 948 A, published Sep. 15, 1993; see also R. F. Brans et (al., (1983) Proc. Natl. Acad. Sci. USA 80:2077-2080; R. F. Bruns et al., (1986) Mol. Pharmacol. 29:331-346; M. F. Jarvis et al. (1989) J. Pharma. Exp. Therap. 251:888-893; K. A. Jacobson et (al., (1989) J. Med. Chem. 32:1043-1051). The properties of human receptors can be studied using recombinant human receptors (Robeva A S, Woodard R, Jin X, Gao Z, Bhattacharya S, Taylor H E, Rosin D L and Linden J (1996), "Molecular characterization of recombinant human adenosine receptors," Drug Dev Res 39: 243-252.; Salvatore C A, Jacobson M A, Taylor H E, Linden J and Johnson R G (1993), "Molecular cloning and characterization of the human A3 adenosine receptor," Proc Natl Acad Sci USA 90:10365-10369.)
8-Phenylxanthines, methods of their synthesis and their use in human and veterinary therapy for conditions associated with the cell surface effects of adenosine have been described (EP 0 203 721, published Dec. 3, 1986). However, this publication is silent as to adenosine receptor subtypes and subtype specificity of disclosed compounds. In WO 90/00056, a group of 1,3-unsymmetrical straight chain alkyl-substituted 8-phenylxanthines were described as being potent bronchodilators. This disclosure is likewise silent as to the receptor subtype specificity of disclosed compounds.
Methods of treating conditions related to the physiological action of adenosine have, to date, proven inferior due to the presence of multiple subtypes present in the animal tissue utilized (R. F. Bruns et al., (1986) Mol. Pharm. 29:331-346) and the differences between species in the affinity for adenosine analogs and the physiological effects of adenosine (Ukera et al., (1986) FEBS Lett, 209:122-128).